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Abstract

An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump–Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420–2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level.

Fig. 2 PA Raman signals in liquids obtained with parallel polarization of the pump and Stokes beams. (a) PA Raman spectra (PARS) of acetone obtained in a thin (120 μm) microscope slide by scanning of first beam in the visible spectral range (PARSVIS) and in the NIR range (PARSNIR) at a fixed second beam wavelength of 639 nm (15,649 cm–1). Dash curves show linear PA background during spectral scanning of first beam alone (i.e., without second beam at 639 nm). (b) PA Raman spectra of benzene obtained in a microscope slide at a fixed pump wavelength of 639 nm and spectral scanning of a Stokes beam in the NIR range. (c) PA signal amplitude as a function of delay time between the pump and Stokes beams for acetone. (d) PA Raman signal for acetone at delays of 0 (top, left) and 20 ns (top, right), compared to signals induced by the pump (bottom, left) and the Stokes (bottom, right) beams alone. Amplitude and time scale: 10 mV/div and 1 μs/div.

Fig. 3 PA Raman signals in olive droplets in a cuvette. (a) PARS at pump and Stokes beam frequencies in the visible and NIR ranges obtained by scanning at a pump beam wavelength tuned in the visible range and at a fixed Stokes wavelength and by scanning at a fixed pump beam wavelength and at a Stokes beam wavelength in the NIR range. (b) PA Raman signal amplitude as a function of the pump laser energy at 529 nm. (c,d) PA signal amplitude as function of delay time between pump and Stokes pulses with pump wavelengths in the visible (c) and the NIR ranges (d). (e) PT Raman signal at a νP of 539 nm and a νS of 639 nm at delays of 0 (left) and 20 ns (right). (f) Linear PA signals at delays of 2.5 μs (left), as well as with a 1-mm distance between pump and Stokes beams (right). Amplitude and time scales: 50 mV/div and 1 μs/div.

Fig. 6 PA/PT detection of adipocytes in vivo in a mouse mesentery model. (a) Mouse mesentery with single layer of adipocytes and the He-Ne probe beam. (b) PT Raman image of a single adipocyte in vivo using two wavelengths: 835 nm as pump (30 µJ) and 639 nm (25 µJ) as Stokes wave. (c) Conventional PT image of a single adipocyte in vivo using a 550 nm (50 µJ) for heating of cellular cytochromes, and 639 nm, as probe beam (10 nJ) at 30-ns delay [26]. (d) PA Raman signals from adipocytes in vitro at delays of 0 (left) and 20 ns (ritgh). (e) PA Raman signals from adipocytes in vivo at delays of 0 (left) and 20 ns (right). The signal oscillations are associated with reflections of acoustic waves in the cuvette. The time scales: 1μs/div (d), and 10 μs/div (e).